All enveloped viruses enter cells by the process of membrane fusion, and produce enveloped progeny virus by budding through a cellular membrane. Understanding virus membrane fusion and budding at the molecular level will provide critical insights into viral disease mechanisms, the development of anti-viral therapies, and the membrane fusion and budding reactions of eukaryotic cells. Semliki Forest virus (SFV) is a well-characterized alphavirus that infects cells by low pH-triggered membrane fusion and buds through the cell plasma membrane. Membrane fusion is mediated by the SFV E1 glycoprotein through a defined series of low pH-dependent conformational changes, and is strongly dependent on cholesterol and sphingolipid in the target membrane. During budding, E1 forms an icosahedral lattice that organizes the structure of the new virus particle. Interestingly, budding is also promoted by cholesterol in the host cell membrane. The overall goal of this grant is to define the molecular features of the entry and exit of alphaviruses from cells, and the role of cholesterol and sphingolipid in these processes. We have isolated and characterized three SFV mutants (srf-3, -4, & -5) that are strikingly increased in their ability to both enter and exit from cholesterol-depleted cells. Each mutant has a single amino acid substitution in E1, srf-3/P226S, srf-4/L44F, srf-5N178A. Both srf-4 and srf-5 are also sphingolipid-independent for fusion. We will characterize the roles of the 3 distinct E1 regions identified by these mutations, using in vitro mutagenesis to define the specific residues and protein interactions involved, and assays of fusion and E1 conformational changes to determine the functional effects of the mutations. Our recent studies indicate that the E1 fusion peptide strongly associates with membrane rafts following its low pH triggered membrane insertion. Our hypothesis is that this reflects the association of cholesterol with the fusion peptide. We will characterize the properties of E1-raft association and the influence of srf and fusion peptide mutations on association. We will directly assay for E1-cholesterol interactions using photocholesterol and fluorescent probes. We have developed an SFV budding assay based on cell surface biotinylation of the envelope proteins and virus retrieval by streptavidin-conjugated magnetic particles. This system will be used to reconstitute the budding of SFV from broken cells. We will focus on defining the nucleotide and cytosol requirements for budding, and on characterizing the role of cholesterol.